Mendelian randomization study supports effect of gut microflora on fractures

To investigate the possible causal relationship between intestinal microflora and fractures using Mendelian randomization (MR). A 2-sample MR study of gut microbiota and fractures was conducted using a weighted inverse variance analysis with tests for heterogeneity, horizontal pleiotropy, and sensitivity. A causal association between fracture risk and specific bacterial taxa was identified at various taxonomic levels: 2 (Bacteroidia, P = .0304; Deltaproteobacteria P = .0304) at the class level, 3 (Bacteroidales, P = .0428; Desulfovibrionales, P = .0428; Enterobacteriales, P = .0208) at the order level, 2 (FamilyXI, P = .0304; Enterobacteriaceae P = .0332) at the family level, and 1 (Alistipes, P = .0405) at the genus level. This study revealed a causal relationship between gut microflora and fracture risk, demonstrating that the effect of different flora taxa flora abundance on fracture risk differs. It provides a reference for further studies.


Introduction
The human gut is inhabited by thousands of bacteria that affect host physiology through unique mechanisms of action, and these bacterial taxa are collectively referred to as the gut microbiota. [1]icrobiologists and clinicians have increasingly emphasized the clinical value of intestinal microflora in the last decade, and several studies have been conducted to identify the association of intestinal microflora with various human diseases and abnormal physiological mechanisms.For example, the effect of gut microflora on obesity, [2] diabetes, [3] human aging, [4] psychiatric disorders, [5] and cardiovascular diseases, etc. [6] The relationship between the gut microflora and the human body has opened a new chapter.
A fracture, as we all know, is a disruption of the continuity and stability of the bone.It is 1 of the most common diseases in orthopedics and is associated with various factors, mainly external trauma, aging, and bone loss. [7]Recent studies have revealed the association between gut microflora and bone and have further explored the relationship between gut microflora and bone health. [8]An observational study found a possible correlation between gut microflora and fracture risk. [9]The gut microbiota regulates osteoporosis through the "brain-gut-bone" axis, and Guo et al showed that Lactobacillus rhamnosus GG can improve osteoporosis by regulating the T helper cell 17/Treg balance.Osteoporosis is a major risk factor for bone fractures, which may indicate that our gut microflora may play an important role in the occurrence of bone fractures. [10,11]However, no other evidence supports an association between gut microflora and fracture risk.
Mendelian randomization (MR) is a reliable epidemiological method for proving causality using information about genetic variation as an instrumental variable. [12]Its strong evidence-based medical evidence could better demonstrate the association between gut microflora and fracture risk.Genomewide association studies (GWAS) have allowed us to explore the relationship between gut microbiota and fracture risk further.

Study design
This study used 2-sample MR to investigate the possible causal relationship between gut microbiota and fracture risk.Approximately 196 gut microbial taxa with unique species names recorded in MiBioGen were used as exposures.About 10 Fracture information from FinnGen includes forearm fractures, lower leg fractures including ankle, shoulder and upper arm fractures, lumbar spine and pelvis fractures, wrist and hand Gut microbiota data were obtained from MiBioGen (https:// mibiogen.gcc.rug.nl/), which included 340,024 individuals from 18 cohorts, most of European ancestry (n = 13,266), examined 211 gut microbiota abundances, and recorded a total of 122,110 associated single nucleotide polymorphisms (SNPs) (15 unnamed taxa were excluded from this study.).All 10 outcome variables related to fractures were obtained from the FinnGen Biobank (Round 9) (https://r9.finngen.fi/),a large GWAS study based on the Finnish biobank Project, with sample sizes and information on nSNPs shown in the table below (Table 1).The diagnostic classifications of fractures were based on Iinternational Classification of diseases-10, and the present study defined the phenotypes (Table 1).
The data are all from the European population but from different countries, so the outcomes are relatively independent of the exposed GWAS data, and there is no significant sample overlap.
The data used in this study were obtained from publicly available GWAS, with links provided for inclusion in the text, and therefore did not require ethical review.

Filtering of instrumental variables
We used P = 1e −5 as a threshold to screen relevant SNPs as possible instrumental variables (IV), thus ensuring a solid correlation of IV.To remove the interference between SNPs due to linkage disequilibrium, clump analysis was performed, and populations with nSNPs < 3 were excluded, thus ensuring independence between the final IV SNPs.Finally, the F-statistic was calculated with the formula F = beta2/se2; the SNPs with F-statistic < 10 were excluded, and reliable SNPs were obtained as IV. [13]

Data analysis
All statistical analyses and plots were performed in Rstudio software based on R4.3.1, and the main R package used was "TwosampleMR." A 2-sample MR method was used, with inverse variance weighted as the primary method and MR-Egger and Weighted Median as supplements for MR analysis.Cochrane's Q test, leave-one-out test, and MR-Egger intercept test were used to perform heterogeneity tests, sensitivity analyses, and tests of horizontal multiple validity.When there is horizontal multiplicity, the analytical discussion is meaningless.When there is no horizontal pleiotropy and no heterogeneity, a fixed effects model is used to represent and judge the results.When there is no horizontal pleiotropy and heterogeneity, the randomeffects model represents and judges the results. [14]To reduce the occurrence of type II error, the P value of the inverse variance weighted (IVW) results were corrected hierarchically from the phylum-family-genus-species level of the intestinal microflora using the Benjamini-Hochberg method. [15]he results screened for the absence of pleiotropy, and the corrected P value was still less than the 0.05 threshold, which was used as the result.The heterogeneity of the results was again checked using the MR-PRESSO Global test, and depending on the heterogeneity of the results, either a fixed-effects model or a random-effects model was selected to represent the final result.The positive results were summarized and presented.

Results
Excluding results with horizontal pleiotropy and selecting the type of IVW method used based on heterogeneity, we finally found 2 taxa of bacteria, Bacteroidia and Deltaproteobacteria, associated with the risk of fracture occurrence at the class level and 3 taxa of bacteria, Bacteroidales, Desulfovibrionales, and Enterobacteriales, were associated with fracture risk at the order level, Desulfovibrionales and Enterobacteriales were associated with fracture risk at the order level, 2 taxa of bacteria, FamilyXI and Enterobacteriaceae, were associated with fracture risk at the family level, and Alistipes was associated with fracture risk at the genus level (Figs. 1 and 2).
It was tested that none of the above positive results were heterogeneous with horizontal pleiotropy, so the fixed effects model was chosen to explain the results.After performing the leaveone-out test, the results were stable and reliable (Figs. 2 and 3).
In MR analysis at the Class level, we see that Bacteroidia [IVW (fixed effects): P-adjust = 0.0304, OR (95%CI) = 0.837 (0.743~0.943)],Deltaproteobacteria [IVW (fixed effects): P-adjust = 0.0304, OR (95%CI) = 0.846 (0.755~0.947)], both taxa of Bacteroidia were causally associated with Fracture of forearm, with a 16.3% decrease in the risk of Fracture of forearm occurring with each 1 standard deviation increase in the abundance of Bacteroides.The risk of Fracture of the forearm decreased by 15.4% with each standard deviation increase in the abundance of Deltaproteobacteria.
In the order level MR analysis, we see that Bacteroidales [IVW (fixed effects): P-adjust = 0.0428, OR (95%CI) = 0.837 (0.743~0.943)],Desulfovibrionales [IVW (fixed effects): P-adjust = 0.0428, OR (95%CI) = 0.843 (0.749~0.948)] were causally associated with a forearm fracture, Enterobacteriales [IVW (fixed effects): P-adjust = 0.0208, OR (95%CI) = 0.662 (0.518~0.847)] were causally associated with a lumbar spine fracture, and pelvis was causally associated.The results suggest that with each standard deviation increase in the abundance of Bacteroidia's flora, we have a 16.3% decrease in the risk of occurrence of fracture of the forearm.For each standard deviation increase in the abundance of Desulfovibrionales, the risk of forearm fracture decreased by 15.7%.For each standard deviation increase in the abundance of Enterobacteriales, the forearm fracture risk was reduced by 33.8%.
In the MR analysis at the family level, we see that FamilyXI [IVW (fixed effects): P-adjust = 0.0304, OR (95%CI) = 0.897 (0.844~0.953)] was causally associated with forearm fracture and Enterobacteriaceae [IVW (fixed effects): P-adjust = 0.0332, OR (95%CI) = 0.662 (0.518~0.847)] was causally related to lumbar and pelvic fracture.The results suggest that we decreased the risk of forearm fracture occurrence by 103% with each standard deviation increase in the abundance of the flora of FamilyXI.Lumbar spine and fracture risk decreased by 33.8% with each standard deviation increase in the population abundance of Enterobacteriaceae.
At the genus level, we found only a causal association between Alistipes [IVW (fixed effects): P-adjust = 0.0405, OR (95% CI) = 1.346 (1.144-1.584)]and fracture of the shoulder and upper arm.The results suggest that the shoulder and upper arm fracture risk increased by 34.6% with each standard deviation increase in the abundance of Alistipes flora.
Detailed analysis results, data used, code, specific IV, and other images that may be required can be obtained by contacting the author at ningq14@foxmail.com.

Discussion
Based on the published GWAS study, this study performed a 2-sample MR analysis on 10 exposures of selected gut microbiota and surrogate fractures.The results showed that (1) there was a causal relationship between gut microbiota and fracture.(2) The effect of gut microbiota on fracture was mainly protective but also a risk factor.(3) The abundance of the genus Alistipes flora was a risk factor for fracture.(4) class Bacteroidia, class Deltaproteobacteria, order Bacteroidales, order Desulfovibrionales, order Enterobacteriale, family Enterobacteriaceae flora abundance as a protective factor for fracture (Fig. 3).
Previous studies have also examined gastrointestinal microflora and fracture risk.For example, a cohort study from Japan involving 38 postmenopausal women found an increased fracture risk in a cohort with a lower abundance of the genus Bacteroidales, corroborating our findings. [9]he exact mechanism of the effect of gut microflora on fracture risk is unclear.The impact on bone mineral density (BMD) may influence fracture risk through its effect on BMD.Many studies have reported the impact of gut microflora on BMD, suggesting that our gut microflora may increase the risk of fragility fractures by affecting inflammatory cytokines that lead to bone loss through nutrient absorption. [16]ut microflora can affect BMD from an endocrine perspective and an immune response perspective through the "microbe-gut-bone" axis.[19] Gut microflora can also influence the regulation of immune-inflammatory cells such as T helper cell 17, CD4-positive T-lymphocytes, and Treg cells, activating the corresponding pathways and ultimately causing a chronic inflammatory response that affects bone mineral density and fracture risk. [8,20,21]Gut microorganisms can also influence critical miRNAs in osteoblasts' transcriptional process, thus affecting osteogenesis's operation, such as the effect on miRNA-33-5p, which may affect the differentiation of osteoblasts. [22]bservational studies have also shown that the abundance of Bacteroides and Roseburia in the intestinal microflora of osteoporotic patients is often reduced, as suggested by studies of the intestinal microflora of osteoporotic patients. [23,24]The present research indicates that a decrease in the abundance of Bacteroidales may increase fractures, confirming that gut microflora may influence fracture risk through a pathway that affects bone mineral density.
The effect of gut microflora on related vitamins may also be associated with fracture risk.Members of the gut microbiota are known to synthesize vitamin K and most water-soluble B vitamins, including biotin, cobalamin, folic acid, niacin, pantothenic acid, pyridoxine, riboflavin, and thiamin. [25]For example, vitamin K is a potential protective component against fractures, and the gut microflora synthesizes vitamin K, affecting fracture risk. [26]Supplementation with vitamin B12 and folic acid has been shown to reduce the risk of fracture, while gut microflora can synthesize B12 and thus influence the risk of fracture. [27]ore possible mechanisms require more basic experimental and clinical studies.
This study used the more comprehensive GWAS study on intestinal microorganisms and a MR study with a newer and more complete disease database with a sufficient sample size to enhance the study's credibility.The Benjamini-Hochberg method was used to correct results at the phylum, family, and species levels to reduce the effect of Type II errors.The exposure  and outcome of the both for people of European ancestry, and the 2 samples were relatively independent, which increased the reliability of the study results and eliminated the confounding effect of population differences.
This study also has the limitation that the taxonomic subtypes must be more complete to investigate the causal relationship between gut microbiota and fracture subtypes further.Second, the study population was of European origin, which may limit the applicability of the results of this study.Furthermore, the results of MR studies can only establish causal relationships between exposure and outcome but cannot further investigate the biological mechanisms of gut microflora and fracture.More observational and animal studies are needed to explore this further.

Conclusion
This study reveals a causal relationship between gut microflora and fracture risk and shows that the effect of flora abundance on fracture risk varies by flora taxa.The balance of gut microflora has value in reducing fracture risk.Further related studies can be refined based on the findings of this study so that the theoretical results can be translated into practice.

Figure 1 .
Figure 1.Sensitivity analysis of positive results.

Figure 2 .
Figure 2. Forest plot of positive results of Mendelian randomization analysis.

Figure 3 .
Figure 3. Schematic representation of the effect of gut microflora on fracture risk at different sites.

Table 1
Outcome information.